The abnormal function of beta1 integrin receptors plays a key role in the development of inflammatory diseases such as rheumatoid arthritis. In consequence, intense investigation has centered on defining the molecular pathways that regulate integrin activity. In many instances, changes in integrin function arise from altered integrin affinity for ligand, rather than from changes in receptor expression. Recent studies have suggested that intracellular signaling molecules, such as ras, can modulate integrin conformation, leading to altered cell adhesiveness and/or motility. The molecular effectors of ras that direct conformational changes in beta1 integrins have not been well-defined. Data presented as part of this research proposal supports a role for ras in the regulation of beta1 integrins. The ectopic expression of a dominant negative ras isoform (N17ras) in epithelial cells induces aberrant glycosylation of beta1 integrins. Correspondingly, cells that express aberrantly glycosylated beta1 integrins display numerous deficits in integrin-mediated cellular responses including cell attachment to matrix, cell spreading, focal adhesion formation, cell motility and tyrosine-kinase mediated signal transduction. A central hypothesis is thus proposed that ras regulates the Golgi -mediated glycosylation of beta1 integrins and that, in turn, modifications in glycosylation directly affect integrin function. This hypothesis encompasses three principal areas of inquiry. First, what is the molecular mechanism by which alterations in ras activity induce changes in integrin glycosylation? In Aim l, lectin affinity blotting and carbohydrate sequencing will be used to define the N17ras-mediated changes in the carbohydrate composition of beta1 integrins. Such experiments will likely identify Golgi glycosyltransferases that are candidates for regulation by ras, thus elucidating the molecular events involved in ras-dependent integrin glycosylation. Secondly, do changes in integrin glycosylation directly affect function? In Aim 2, methods will be developed that will allow the expression of N17ras to be uncoupled from the expression of variant integrin glycoforms. These experiments are expected to confirm that differences in glycosylation, rather than other downstream effects of NI 7ras, are directly responsible for deficiencies in integrin signaling. Finally, are rasmediated changes in in te grin glycosylation and function involved in inflammation? In Aim 3, N17ras will be introduced into T lymphocyte and monocytic cell lines in order to determine if, similar to epithelial cells, changes in ras activity can modulate integrin glycosylation and function. The beta1 integrins of T cells and monocytes represent good candidates for regulation by glycosylation because these cells are known to express variant integrin glycoforms in vivo. Moreover, the expression of these variant glycoforms correlates well with changes in cell phenotype. For example, modifications in Golgi--mediated glycosylation of beta1 integrins are observed during both T cell maturation and monocyte activation. Collectively, the experiments proposed in Specific Aims 1-3 are expected to define and characterize a novel, ras dependent, signal transduction pathway that modulates integrin function by regulating the activity of selected Golgi glycosyltransferases. The elucidation of such a pathway may provide insight into the physiologic function of variant integrin glycoforms that are expressed during T cell maturation, monocyte and keratinocyte activation, and metastasis.